The present invention, in some embodiments thereof, relates to articles, compositions and methods for reducing or preventing oxidative stress-associated cellular or macromolecular damage, such as tobacco-induced damage.
The deleterious effects of tobacco abuse are well known. Tobacco is a worldwide public health hazard accounting for significant morbidity and mortality. Although smoking places an abundant oxidant insult to the oropharynx and respiratory tract, the oxidant burden associated with any tobacco consumption (as described hereinbelow) is deleterious to the entire body of the tobacco consumer.
Tobacco consumption leads to development or enhancement of atherosclerosis, cardiovascular diseases, chronic obstructive pulmonary disease, lung cancer, as well as other forms of cancer and peripheral vascular diseases.
Cardiovascular disease is the main cause of death due to smoking. Cardiovascular disease can take many forms, depending on which blood vessels are involved. Main forms include coronary thrombosis, which may lead to a heart attack; cerebral thrombosis, which may lead to collapse, stroke and paralysis; affected kidney arteries, which result in high blood pressure or kidney failure; and blockage of the vascular supply to the legs, which may lead to gangrene and amputation.
Tobacco consumers are more likely to get cancer than non-smokers, particularly carcinomas of the mouth, pharynx, esophagus and lung. Other types of cancer associated with tobacco consumption include bladder cancer, cancer of the oesophagus, cancer of the kidneys, cancer of the pancreas and cervical cancer.
Chronic obstructive pulmonary disease (COPD) is a collective term for a group of conditions that involve block of airflow and include, for example: emphysema and chronic bronchitis.
Other risks associated with tobacco consumption include hypertension, fertility problems, severe asthma, retinoic disorders such as macular degeneration and cataracts, ulcers, periodontal diseases, impotence, Diabetes type 2, Back pain, skin ailments such as premature ageing and wrinkling, osteoporosis, earlier menopause, and damaged and/or weakened immune system.
There are two principal ways to consume tobacco: smoking and smoke-less consumption. The latter comes in various forms: snuff, snus and chewing tobacco. Snuff is a fine-grain tobacco that often comes in teabag-like pouches, which users “pinch” or “dip” between their lower lip and gum. Chewing tobacco comes in shredded, twisted, or “bricked” tobacco leaves that users put between their cheek and gum. Whether it is snuff, snus or chewing tobacco, the user consumes the tobacco letting it sit in the mouth and suck on the tobacco juices, spitting often to get rid of the saliva that builds up. This sucking and chewing allows nicotine (a narcotic drug), to be absorbed into the bloodstream through the tissues of the mouth. Smokeless tobacco has a detrimental effect on the oral cavity plus systemic effects from buccal absorption of nicotine and other chemicals.
Evidence shows that cigars as well as cigarettes are highly toxic and addictive. Tobacco smokers have a similar increased risk for oral and laryngeal cancers. Evidence indicates that one cigar generates levels of carcinogenic particles exceeding those generated by three cigarettes. Fumes from cigars are also of greater consequence to secondary smokers. Epidemiologic studies reveal greater frequencies of heart disease, emphysema, and cancers of the mouth and pharynx in cigar smokers when compared to matched non-smokers.
Tobacco, whether smoked or chewed, causes common untoward effects in the oral cavity. Tobacco smoke (TS) has two chances to exert its deleterious effects in the mouth; when it is inhaled by the smoker and on its exit during exhalation.
Over 30,000 new cases of cancer of the oral cavity are diagnosed annually, accounting for 2-4 percents of all new cancers. The great majority of these patients are users of tobacco products.
Oral squamous cell carcinoma (SCC) is the most common malignancy of the head and neck with a worldwide incidence of over 300,000 new cases annually. The disease is characterized by a high rate of morbidity and mortality (approximately 50%) and in this respect is similar to malignant melanoma. The major inducer of oral SCC is exposure to tobacco which is considered to be responsible for 50-90% of cases world-wide [Epstein and Scully, SCD Special Care in Dentistry 1997; 17:120-8; Holleb et al. Textbook of Clinical Oncology. The American Cancer Society, 1991]. As such, the incidence of oral SCC in tobacco smokers is 4-7 times higher than in non-smokers [see, for example, Ko et al. J Oral Pathol Med 1995; 24:450-3].
Various malignancies are particularly associated with smokeless tobacco consumption. These include oral cancer and cancer of the gastrointestinal tract including esophagus and bladder. Leukoplakia, a tobacco induced white patch on the buccal mucosa, as found in smokers, is a localized irritation due to direct contact of smoked or smokeless tobacco and it is directly related to the frequency and years of tobacco abuse. Although leukoplakia is a benign oral lesion, it has a malignant potential.
In addition, tobacco contributes to other oral symptoms or pathologies of the mouth and teeth. Tobacco may cause halitosis, may numb the taste buds, and interfere with the smell and the taste of food. It may stain teeth and contribute to dental caries. Smokers have more dental tartar (calculus) than non-smokers. Tobacco is associated also with destructive periodontal (gum) disease and tooth loss. Acute necrotizing ulcerative gingivitis (“trench mouth”) is a destructive, painful inflammatory condition occurring mainly in tobacco smokers. Swelling of the nasal and sinus membranes has also been associated, purportedly, in individuals who are “allergic” to TS.
Oral submucous fibrosis occurs mainly in India and is a chronic, progressive premalignant condition. The etiology is chronic chewing of tobacco or areca nut or both. The fibrosis results in restriction of mouth opening and involves the palates, tonsillar fossa, buccal mucosa and underlying muscle. Associated with this condition are also oropharyngeal carcinomas, also with a high frequency in India and associated in 70% of cases with chewing tobacco. Smokeless tobacco and areca nut usage is also common in Pakistan, Bangladesh and Java and in these and Indian immigrants to the United States and United Kingdom.
Studies have estimated that TS has over 3,000 different constituents, of which many are toxic, carcinogenic and/or generate free radical species.
Free radicals are atoms or molecules containing an unpaired electron. Oxygen free radicals include the superoxide free radical (.O2−) and the hydroxyl radical (OH.) which, together with hydrogen peroxide (H2O2) and singlet oxygen (1O2), are jointly called reactive oxygen species (ROS). Due to their high reactivity they may lead to chemical modification and impairment of the components of living cells, such as proteins, lipids, carbohydrates and nucleotides.
Tobacco smoke therefore induces oxidative damage to lipids, DNA and proteins, particularly via protein-SH groups as a consequence of containing high levels of both free radicals as well as aldehydes, including acetaldehyde (ethanol), propanol and acrolein, as well as other deleterious molecules.
Most of constituents of TS have been identified in so-called mainstream and side stream TS. The former is that volume of smoke drawn through the mouthpiece of the tobacco product during puffing while side stream smoke is that smoke emitted from the smoldering cigarette in between puffs. Although tar and nicotine are retained in the filter of cigarettes, this applies mainly to mainstream smoke, when comparing filter and non-filter cigarettes. Mainstream smoke emission is also markedly reduced both in low and in ultra low tar yield cigarettes. However, the emissions of toxic and carcinogenic components in side stream smoke are not significantly reduced in filter cigarettes when compared to non-filter counterparts. Thus, side stream smoke is a major contributor to environmental smoke, affecting both the smoker and their non-smoking counterparts, so called secondary smokers.
Tobacco smoke is divided into two phases; tar and gas-phase smoke. Tar contains high concentrations of free radicals. Many tar extracts and oxidants are water-soluble and reduce oxygen to superoxide radical which can dismutate to form the potent oxidant H2O2. Oxidants in gas-phase smoke are reactive carbon- and oxygen-centered radicals with extremely short half lives.
Cells subjected to oxidative stress develop severely affected cellular function and suffer damage to membrane lipids, to proteins, to cytoskeletal structures and to DNA. Free radical damage to DNA has been measured as formation of single-strand breaks, double-strand breaks and chromosomal aberrations. Cells exposed to ionizing radiation and TS have also been demonstrated to have an increased intracellular DNA damage, a precursor of mutations and development of malignancies. It has been shown that TS elicits protein carbonylation in plasma and that, in contrast, exposure of human plasma to gas-phase but not to whole TS produces oxidative damage to lipids.
Glutathione, a sulfur-containing tripeptide (L-glutamyl-1-cysteine-glycine) is the most abundant non-protein thiol in mammalian cells and is recognized as the primordial antioxidant. Glutathione, in its reduced form, “GSH”, acts as a substrate for glutathione-S-transferase and glutathione peroxidase, enzymes catalyzing reactions involved in detoxification of xenobiotic compounds and in antioxidation of ROS and other free radicals. This ubiquitous protein plays a vital function in maintaining the integrity of free radical sensitive cellular components. Under states of GSH depletion, including malnutrition and severe oxidative stress, cells may then become injured from excess free radical damage and die.
The translocator protein (TSPO) receptor, formerly known as the peripheral-type benzodiazepine receptor (PBR), is an 18-kDa protein located primarily in the outer mitochondrial membrane. TSPO is also referred to in the art as mitochondrial diazepam binding inhibitor (DBI) receptor complex, PK11195-binding sites, isoquinoline-binding protein (IBP), benzodiazepine receptor peripheral, pk18 and ω3 receptor. TSPO receptor is one of a group of proteins that compose the mitochondrial permeability transition pore (MPTP) complex. A background art schematic illustration of MPTP and the pathways associated therewith is presented in FIG. 1.
The MPTP complex of proteins play a central role in various physiological and pathological processes including apoptosis, ischemia, regulation of the mitochondrial membrane potential, mitochondrial respiration, steroidogenesis, immune responses of the cardiovascular system, cell proliferation, and cancer. Other known proteins of the MPTP complex are the 32-kDa voltage dependent anion channel (VDAC) and the 30-kDa adenine nucleotide transporter (ANT). A number of TSPO molecules can be linked to one VDAC molecule. The TSPO receptor can also function without interacting with VDAC and ANT. Additional molecules, such as pk10 (protein of 10 kDa), PBR associated protein1 (PRAX-1) and PBR associated protein 7 (PAP7) can be linked to the TSPO receptor.
TSPO receptor plays an important role in enhancing the ability of cells and tissues to manage ROS-induced damage. Moreover, astrocytes may increase their TSPO expression in hyperammonemia in an attempt to suppress or defend against hyperammonemia associated with increase in ROS production. In addition, TSPO is preferentially expressed in superficial keratinocytes of the differentiated layers of normal epidermis. This preferential expression may be linked to a mechanism of skin protection against ROS damage generated by ultraviolet exposure, as TSPO reduces production of ROS and apoptosis induced by ultraviolet light.
WO 2008/073130 teaches methods for reducing symptoms of conditions whose activity is mediated by TSPO and the cannabinoid CB2 receptors, including inhibition of anxiety, of growth of cancer cells expressing TSPO receptors, and reducing oxygen radical damage to cells, by contacting the cells with the TSPO and the cannabinoid CB2 receptors agonist cis-epoxyeicosatrienoic acid, an inhibitor of soluble epoxide hydrolase (sEH), or both.
Several ligands of TSPO are known. These include benzodiazepine derivatives [e.g. diazepam and 4-chlorodiazepam (Ro5 4864)], isoquinolone carboxamide derivatives (e.g. PK 11195), 2-aryl-3-indoleacetamide derivatives (e.g. FGIN-1), pyrrolobenzoxazepine derivatives, phenoxyphenyl-acetamide derivatives, pyridazinoindole derivatives and 8-oxodihydropurine derivatives. Interestingly, the TSPO does not bind the central benzodiazepine receptor ligand clonazepam. In addition to these high affinity ligands, dicarboxylic porphyrins also bind to the TSPO protein and, although they have lower affinity, it has been proposed that they serve as endogenous ligands (e.g. protoporphyrin IX and hemoglobin). Other endogenous ligands of the TSPO protein are the 86 amino acid polypeptide—endozepine, found in central and peripheral tissue, diazepam-binding inhibitor and its metabolite triakontatetraneuropeptide (TTN), PLA2 and cholesterol. The high affinity binding of cholesterol to TSPO is essential for the process of steroid and bile salt biosynthesis and the low affinity binding of porphyrin is important for the process of heme biosynthesis.
TSPO has been found to be involved in different pathological conditions such as ischemia-reperfusion injury, brain injury, a certain form of epilepsy, neurodegenerative disorders (e.g. Alzheimer's disease, Parkinson's disease), peripheral neuropathy, psychiatric disorders (e.g. anxiety, posttraumatic stress disorder, schizophrenia) and cancer.
Benzodiazepines (BZ's) are a class of lipophilic molecules comprising a benzene ring. To date, there are more then 20 benzodiazepines in clinical use having hypnotic, sedative, anxiolytic, anticonvulsant, muscle relaxant and amnesic properties. These effects are mediated via the central benzodiazepine receptor (CBR). Benzodiazepines are positive allosteric modulators of GABA and bind to a specific subunit of the GABAA receptor [also called the central benzodiazepine receptor (CBR)] and activate it, leading to decreased neurological activity. In order for GABAA receptors to be sensitive to the action of benzodiazepines, they need to contain both an α and a γ subunit. Some BZs, such as diazepam, also binds to the TSPO receptor in peripheral tissues.
Diazepam (Valium) is a benzodiazepine derivative drug that possesses anxiolytic, anticonvulsant, sedative, skeletal muscle relaxant and amnestic properties. It is commonly used for treating anxiety, insomnia, seizures, alcohol withdrawal, and muscle spasms.
TSPO receptor ligands have been described as therapeutically active agents for various uses.
For example, U.S. Pat. No. 7,220,739 teaches methods for treating a condition associated with disregulation of the process of cell death in a subject, comprising administrating to the subject an effective amount of a benzodiazepine compound. The benzodiazepines taught in this patent are identified by their inability to bind to CBR and by low affinity to TSPO. The conditions associated with deregulation of the process of cell death taught in this patent include autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, graft-versus host disease, and myasthenia gravis; chronic inflammatory conditions such as psoriasis, asthma and Chrones disease; hyperproliferative disorders of neoplasm's such as B-cell or T-cell lymphoma; viral infection and other conditions such as osteoarthritis and atherosclerosis. According to the teachings of U.S. Pat. No. 7,220,739, the therapeutic potential of benzodiazepines is due to their proapoptotic and cytotoxic properties.
Sakai et al. [European Journal of Pharmacology 2006; 550:8-14] describe cell proliferation inducing properties of TSPO ligands in tumor cells. Sakai et al. suggest that discrepancies regarding the observed role of TSPO ligands in cell proliferation, seem to rely on the doses of TSPO ligands used, wherein subjecting the cells to low concentrations of TSPO ligands (nM) leads to an increase in cell proliferation whereas high concentration (μM) inhibits cell proliferation.
The present inventors have recently synthesized ligands of the TSPO receptor for the treatment and prevention of brain damage due to traumatic brain injury (TBI) and for the treatment and prevention of neurodegenerative diseases (see, WO 2008/023357). The compounds taught in WO 2008/023357 are quinoxaline, quinazoline and phthalazine derivatives, as well as multimeric compounds. According to the teachings of WO 2008/023357, the compounds described therein bind to TSPO, reduce basal apoptotic levels in neuronal cells, as well as apoptosis induced by glutamate. Glutamate is known as an important agent which causes secondary brain damage after traumatic brain damages, and is also involved in neurodegenerative diseases.
U.S. Pat. No. 5,550,124 teaches agonists of TSPO for the prevention and treatment of various central nervous system (CNS) injuries. This patent also teaches methods of screening for new TSPO agonists.
U.S. Pat. No. 5,776,946 teaches the use of compounds which bind with high affinity to TSPO receptor as anti-inflammatory agents. Such compounds include isoquinoline and benzodiazepine derivatives. Inflammatory conditions treatable by these compounds, as taught by this patent, include rheumatoid arthritis, lupus erythematosus, Sjogren's syndrome, osteoarthritis, multiple sclerosis, Behcet's disease, temporal arthritis and dementia of the Alzheimer type.
U.S. Pat. No. 6,686,354 teaches use of agonists and antagonists of TSPO to induce or inhibit cardiac hypertrophy. In particular, this patent teaches use of antagonists of TSPO in the prevention or treatment of decompensated cardiac hypertrophy and eventually, heart failure. U.S. Pat. No. 6,686,354 also teaches the use of TSPO agonists in the management of conditions calling for increased blood flow or cardiac output such as injury or functional compromise, of the heart, increased demand for physical exercise by athletes or by those who need extra help to improve cardiac performance as a result of a disability, existing atrio-ventricular (A-V) shunts, an acquired or inherited predisposition to cardiac contractile protein dysfunction.
U.S. Pat. No. 7,267,977 describes the involvement of TSPO in human breast cancer, wherein the invasive and metastatic ability of human breast tumor cells is proportional to the level of TSPO expressed and correlates with the subcellular localization of TSPO in these cells. According to the teachings of this patent, TSPO is found primarily in the nuclei of aggressive tumor cells whereas TSPO is found primarily in the cytoplasm of invasive but non-aggressive cells. U.S. Pat. No. 7,267,977 teaches methods for detecting the level of metastatic ability of cells by measuring the level of TSPO in tumor cells as compared to normal cells. U.S. Pat. No. 7,267,977 also teaches methods and compositions effective for reducing or inhibiting TSPO expression or function, for use as a treatment for metastatic breast cancer. The compositions taught by this patent comprise antibodies or compounds capable of reducing or inhibiting TSPO such as TSPO antagonists as well as various ions, anions, phospholipids and factors which affect the stability of the TSPO receptor.
Other suggested therapeutical applications for TSPO receptor ligands include topical use in the treatment of skin (cutaneous) stress (see, U.S. Pat. No. 6,767,533) and treatment of neuropathic pain (see, EP patent application No. 06732484.8, International publication No. WO 2006/115302).
U.S. Pat. No. 6,379,649 teaches imidazo(1,2-a)pyridines and related compounds, for the treatment and detection of disorders that are characterized by an abnormal density of TSPO receptors such as neurodegenerative disorders and tumors.
Several prior art approaches have been employed in order to reduce or prevent incidence of oral disease resulting from oxidant injury.
For example, cigarette filters are used to trap TS tar but do not affect the gas-phase compounds.
One approach has employed a filter for TS providing chemoabsorptive properties to reduce aldehyde concentration in TS (see, U.S. Pat. No. 5,060,672).
Another approach has employed oral megadoses of antioxidants in attempts to reduce generation of H2O2 resulting from the “respiratory burst” reaction associated with phagocytic activity of macrophages and neutrophils. It has been shown that smokers have a higher “respiratory burst” reaction than non-smokers and that this may be associated with the increased incidence of aerodigestive tract disease in the former.
In yet another approach, dipeptide compounds with pharmaceutical properties to increase glutathione levels were employed (see, for example, U.S. Pat. No. 4,761,399).
A further approach utilized a glycine carboxylic acid alkyl mono-ester of glutathione to increase cellular GSH levels (see, for example, U.S. Pat. No. 4,710,489).
In yet a further approach, administration of a combination of glutathione and selenium was suggested for preventing oxidant injury resulting from exposure to TS (see, for example, U.S. Pat. No. 5,922,346).
In another approach, administration of a combination of glutathione, ascorbic acid, selenium and a sulfur-containing amino acid was suggested in order to prevent oral oxidant injury (see, for example, U.S. Pat. No. 6,228,347).
In yet another approach, administration of a combination including some or all of the following antioxidants; L-glutathione, L-selenomethionine, L-selenocysteine, ascorbyl palmitate, ascorbic acid esters, L-cysteine, N-acetyl-1-cysteine, tocopherol acetate, tocopherol succinate, vitamin A, a zinc salt, methionine and taurine was suggested in order to provide intra-oral protection from oxidant injury (see, U.S. Pat. No. 5,829,449).
The present inventors have previously described novel smoking filters and oral compositions for reducing tobacco associated damage in the aerodigestive tract (see, U.S. Pat. No. 6,789,546, which is incorporated by reference as if fully set forth herein). These compositions include active agents which are capable of reducing or preventing tobacco associated loss of peroxidase activity in the aerodigestive tract.
U.S. Pat. No. 5,922,346 teaches a composition for reducing free radical damage induced by tobacco products and environmental pollutants comprising, as active ingredients, reduced glutathione and a source of selenium selected from the group consisting of elemental selenium, selenomethionine and selenocysteine, the active ingredients being combined with suitable carriers and flavorings for their intra-oral administration as gels, lozenges, tablets and gums in concentrations for reducing free radical damage induced by tobacco products and other environmental pollutants to the oral cavity, pharynx and upper respiratory tract of a user and secondary smokers.
U.S. Pat. No. 5,906,811 teaches a method for reducing free radical damage induced by tobacco products and environmental pollutants comprising administering in a suitable carrier in concentrations for effectively reducing said free radical damage to the oro-pharynx and upper respiratory tract of a user a combination of from 0.01 and 10% (weight) glutathione, from 1.0 to 25% (weight) ascorbic acid, from 0.001 to 10% (weight) of a source of selenium and from 0.001 to 2.0% (weight) of a sulfur containing amino acid.
These aforementioned attempts to reduce tobacco damage are used as an adjuvant treatment following or prior to tobacco consumption, but not concomitantly with tobacco consumption.
U.S. Pat. No. 6,138,683 teaches a composition for inclusion within a quantity of smokeless tobacco, selected from the group consisting of chewing tobacco and snuff, for reducing free radical induced damage to the oro-pharyngeal cavity of the user, said composition comprising L-glutathione and a source of selenium in combination with said smokeless tobacco.
PCT/IL2008/000101, by the present assignee, describes methods, pharmaceutical compositions, oral compositions, filters and tobacco products for preventing or reducing tobacco smoke-associated injury in the aerodigestive tract of a subject, which can be used to prevent or reduce loss of OPO activity or CN−, redox-active metal ion- or aldehyde-induced cell death resulting from TS-associated oxidative stress. Some of the agents described in this document are CN− chelators and iron chelators.
PCT/IL2008/000628, by the present inventors, describes compositions, articles and methods comprising copper chelating agents such as, for example, penicillamine, as well as structural analogs thereof, for the treatment of tobacco-associated damage.